Similar AlbeitNot the Same: In-Depth Analysis ofProteoforms of Human Serum Bovine Serum and Recombinant Human Fetuin

摘要

Fetuin, also known as alpha-2-Heremans Schmid glycoprotein (AHSG), belongs to some of the most abundant glycoproteins secreted into the bloodstream. In blood, fetuins exhibit functions as carriers of metals and small molecules. Bovine fetuin, which harbors 3 N-glycosylation sites and a suggested half dozen O-glycosylation sites, has been used often as a model glycoprotein to test novel analytical workflows in glycoproteomics. Here we characterize and compare fetuin in depth, using protein from three different biological sources: human serum, bovine serum, and recombinant human fetuin expressed in HEK-293 cells, with the aim to elucidate similarities and differences between these proteins and the post-translational modifications they harbor. Combining data from high-resolution native mass spectrometry and glycopeptide centric LC-MS analysis, we qualitatively and quantitatively gather information on fetuin protein maturation, N-glycosylation, O-glycosylation, and phosphorylation. We provide direct experimental evidence that both the human serum and part of the recombinant proteins are processedinto two chains (A and B) connected by a single interchain disulfidebridge, whereas bovine fetuin remains a single-chain protein. Althoughtwo N-glycosylation sites, one O-glycosylation site, and a phosphorylationsite are conserved from bovine to human, the stoichiometry of themodifications and the specific glycoforms they harbor are quite distinct.Comparing serum and recombinant human fetuin, we observe that theserum protein harbors a much simpler proteoform profile, indicatingthat the recombinant protein is not ideally engineered to mimic humanserum fetuin. Comparing the proteoform profile and post-translationalmodifications of human and bovine serum fetuin, we observe that, althoughthe gene structures of these two proteins are alike, they representquite distinct proteins when their glycoproteoform profile is alsotaken into consideration.